Polymyxin production



Patented Oct. 16, 1951 UNITED STATES PATENT OFFICE POLYMYXIN PRODUCTION Robert G. Benedict, Peoria, 111., assignor to the United States of America as represented Secretary of Agriculture by the No Drawing. Application May 7, 1948, Serial No. 25,775

(01.195-96) (Granted under the act of March 3, 1883, as

6 Claims.

amended April 30, 1928; 370 0. G. 757) 1947, now abandoned, by Benedict and Stodola,

a method for producing polymyxin is described. This method comprises cultivating B. polymyra in an aqueous medium comprising a proteinaceous material and an assimilable carbon source. The polymyxin is elaborated in a highly active form and may be isolated from the culture medium. The antibiotic thus produced is effective in A preventing the growth of several pathogenic gram negative organisms. i

This invention relates to the discovery of an organism capable of elaborating much greater amounts of polymyxin, and to a method of cultivat ing the organism. More specifically, this in-.

vention relates to the discovery of a. stable ultraviolet mutant, which gives yields of polymyxin greatly exceeding those heretofore obtained by any other organism.

4. strain of B. polymyxa was subjected to ultrairradiation, during the course of extended investigation. According to a standard procedure, the culture was spread upon an agar surface so as to obtain isolated survivors. The surviving cells were allowed to develop. A' culture from a colony of one of the survivors has been found to produce polymyxin in amounts approximately five times as large as that produced by the parent strain.

This mutant will hereafter be identified as NRRL 8-719 (u. v.-37). -It is stable, retaining its capacity for producing polymyxin at this time beyond the fortieth generation. It is capable of producing greatly increased yields under substantially all fermentation conditions applicable generally to B. polymyzra organisms.

I have also discovered that the yield of the polymyxin from this or other B. polymyma organisms can be greatly increased by feeding" during the fermentation period.

In the past, polymyxin has been produced by cultivating 'B. polymyza according to the description contained in the aforementioned application. The organism is one which notoriously produces a slime or mucilagin'ous material in the course of the cultivation which usually takes up to.five days or longer. The slime production is aggravated by the initial presence of assimilable carbon source, such as sugars or starch. If more than 2 percent assimilable carbon source is employed in the initial fermentation medium, the

- resulting culture liquor is diiilcult to handle and recovery of the desired antibiotic ,isundesirably tedious and involved. I have discovered that about double the ultimate yield of antibiotic may be obtained, and at the same time slime formation reduced considerably by feeding" the. assimliable carbon in the fermentation process. For example, I use media heretofore employed containing initially about 1 to 2 percent sugar and add about an equal amount of sugar during the fermentation, preferably during the fortyeighth to seventy-second hour. Accordingly, the final culture liquors contain very little slime or mucilaginous material, and the increased amount of the antibiotic is easily recoverable.

In general, the'fermentations according to my invention are carried out,under submerged aerobic conditions. The assimilable carbon source employed may be dextrose or glucose, xylose, hyd l, starch, and the like. The proteinaceous material may be com steep liquor, peanut flour, soya fiour, yeast extract, ground grain, or the like, in an amount ranging from one-half to five percent, preferably one-half to one and onehalf percent of the medium. For my purposes, I prefer peanut flour or meal as the proteinaceous material, since it results in high yields of anti.- biotic and is economical.

The following examples exhibit the invention in'greater detail. The parts are by weight unless otherwise specified.

EXAMPLE I.

Aqeous media consisting of 0.5 percent soya' meal from which the oil had beenextracted and 1 percent glucose to which 1 percent calcium:

lated with NRRL 3-719 (u. v.-3'l) were made after 72, 96 and 120 hours fermentation. At the end of '72 hours fermentation. 1 percent glucose was added to the media.

For comparison, identical media were inocu- Assays were made after 68, 92, and 116 hours fermentation. After 68 hours fermentation, 1 percent glucose was added to the flasks.

The results of this example are shown in Table 1. The tabular values are units of the antibiotic produced per ml. of culture liquor. The unit of antibiotic is an arbitrary value which will just inhibit the growth of Escherichia coli on an agar plate. It is approximately equivalent to 0.5 microgram of the purified substance.

Table 1 Assay (units) Organism 72 hours 90 hours 120 hours iermeniermenfermentstion tatlon tatlon NRRL B-719 120 i)! 135 N REL B-367 165 144 210 NBBL B-M so 12s 22s 08 hours 92 hours 116 hours fermeniermenfermentation tstlon tatiou 329 1, a NBBL B-7l0 (u. v.47) m 24 m 768 EXAMPLE II fermentations were carried out employing the original medium, unsupplemented, as a control, and varying the time at which additional glucose was added in the remaining flasks oi the set. The fermentations were carried out in shaker flasks. The results are shown in Table 2.

Table 2 Assay (units) 04 hours BBhours ll2hours 136 hours iermeniermenfermenfermentation tstion tstlon tatlon Organise! .4 Control 324 104 124 1 glucose at 48th hrass 240 ass m l glucose at 72nd hr 344 102 332 I Orsssisss B Control 160 no 176 1 glucose at 48th hr 1m m 360 184 1 glucose at 72nd hr 192 m 400 100 Orssslus C Control 212 B2 164 l gluon. at 7nd hr M4 82 352 212 1 glucose at 88th br 210 148 232 184 The following example illustrates the advantageous properties of peanut meal as the proteinaceous material in the production of the antibiotic.

EXAMPLE III Aqueous media consisting of 1 percent peanut meal from which the oil had been extracted and 1 percent glucose to which 1 percent calcium carbonate had been added, were inoculated with three various known strains of B. polvmuza organisms. The peanut meal contained about 0.00 percent lipids. At the end of about 72 hours of fermentation, an additional 1 percent glucose was added. For comparison, similar fermentations were conducted substituting an equal amount of soya flour for the peanut meal. The results are shown in Table 3.

l Assay taken at 88 hours fermentation.

The peanut meal or flour used should have a low oil content; In general, within the preferred range of proteinaceous material of onehalf to one and one-half percent, the yield of the antibiotic increases with an increase of peanut flour or meal.

Having thus described the invention, what is claimed is:

1. A process for producing polymyxin comprising cultivating B. p ll/mum NRRL 13-719 (u. v.-37) in an aqueous medium comprising a proteinaceous material which medium wntains one to two percent assimilable carbon source at the start of the fermentation. and adding onehalf to two percent supplementary assimilable carbon source during the course of the fermentation.

2. The process of claim 1 in which the supplementary assimilable carbon source is added during the 48th to 72nd hour of fermentation.

3. The process of claim 1 in which the proteinaceous material is peanut flour. the assimilable carbon source is glucose, and the supplement is added during the 48th to 72nd hour of fermentation.

4. A process for producing polymyxin which comprises cultivating B. polumuza NRRL 3-710 (u. v.47) under submerged aerobic conditions in an aqueous medium containing one-half to one and one-half percent protelnaceous material, one to two percent assimilable carbon source and about one percent calcium carbonate, and adding one-halt to two percent additional carbon source during the 48th to 72nd hour of fermentation, and recovering the produced polymyxln.

5. The process of claim 4 in which the proteinaceous material is peanut flour and the carbon source is dextrose.

5 6 6. The process for producing polymyxin which UNITED STATES PATENTS comprises cultivating B. polymyxa NRRL 3-719 Number Name Date (u. v.-37) in an aqueous medium comprising}; 2,344,025 Christensen a Man 14' 1944 proteinaceous material and in the preselgoe of 2,401,778 sjolander June 11' 1946 an assimilable carbon source. 5 2 445 748 Demerec July 27 194 ROBERT G. BENEDICT. 7 OTHER REFERENCES Stansiey et a1.: Bulletin Johns Hopkins Hos- REFERENCES CITED pital. July 1947. pp. 4344.

The following references are of record in the m Br w r J ll Btwt. October 1943, pa es file of this patent: 396. 

1. A PROCESS FOR PRODUCING POLYMYXIN COMPRISING CULTIVATING B. POLYMYXA NNRL B-719 (U. V.-37) IN AN AQUEOUS MEDIUM COMPRISING A PROTEINACEOUS MATERIAL WHICH MEDIUM CONTAINS ONE TO TWO PERCENT ASSIMILABLE CARBON SOURCE AT THE START OF THE FERMENTATION, AND ADDING ONEHALF TO TWO PERCENT SUPPLEMENTARY ASSIMILABLE CARBON SOURCE DURING THE COURSE OF THE FERMENTATION. 